This isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami was described by Carraway and Leeman (J. Biochem. 248, 6854-6861 (1973)). These investigators utilized batches of frozen hypothalamic tissue representing 2,000-4,500 animals (cows) weighing a total of 20-45 kg. The tissue was homogenized to a uniform consistency with an equal volume of -20.degree. acetone 1 N-CHl (100:3 v/v) in a colloid mill. Elaboration of this initial step and final removal of acetone yielded an aqueous residue which was lyophilized. These initial steps were performed on a preparative scale.
Purification utilizing chromatography on a G-25 Sephadex follows. Material from a bioassayed active region was pooled, lyophilized, and then taken up in 0.1 M acetic acid (100 ml/20 kg of hypothalami) and rechromatographed on a 5-liter column kept at room temperature. Again, the active region was pooled and lyophilized. Cation exchange chromatography was next utilized for further purification.
Next, preparative paper electrophoresis was utilized. The active material from 45 kg of hypothalami was applied to a 10-cm band to Whatman No. 3 MM paper and subjected to electrophoresis. Fifty percent of the active peptide was recovered from this electrophoresis and found to be a pure peptide.
By the above steps, the extracted peptide was purified approximately 200,000-fold, and approximately 3-5 nmoles of pure neurotensin was obtained/kg of wet tissue.
This isolated neurotensin from bovine hypothalami induced hypotensin in the rat and stimulated the contraction of guinea pig ileum and rat uterus. It produced relaxation of the rat duodenum. These pharmacological properties classified neurotensin as a "kinin". Its chemical composition distinguished it from any known peptide.
Subsequent to the above described isolation of neurotensin, its amino acid sequence was established by the report of Carraway and Leeman (J. Biochem. 250, 1907-1911 (1975)). The established sequence is that of pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH.
(The nomenclature and symbols used in expressing this sequence follow the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature (J. Biochem. 247, 977 (1972)). The results of enzymic hydrolyses and the specificities of the enzymes used to establish this sequence of neurotensin indicated that all of the amino acids are unsubstituted and are of the L-configuration.
Neurotensin was synthesized by Carraway and Leeman (J. Biochem. 250, 1912 (1975)) provided by a manual solid-phase method by a procedure which is different from the synthetic methodology used for the invention described herein. In their synthesis of neurotensin Carraway and Leeman had as their primary synethetic goal the synthesis of Gln.sup.1 neurotensin which was then treated by heat and acid to cyclize the Gln.sup.1 -moiety to the pGlu.sup.1 -moiety or to neurotensin. In this procedure, their protected peptide was cleaved from the resin using hydrogen bromide and trifluoracetic acid. Subsequent catalytic hydrogenation removed the nitro groups from the two nitroarginyl residues before cyclization of the Gln.sup.1 -moiety to the pGlu.sup.1 -moiety.